RNA quality and quantity for RNA-seq

[Ahmadiut]

Hi
I have five samples and want to send them to an overseas institute for RNA-seq. My samples are from a native plant which RNA extraction of it is very difficult. Here I attached two files that show the agaros gel and nanodrop results. I am not sure about their qual and quant, Could you please tell me your opinions about them?
Thanks in advance

[dpryan]

The brightness of the wells suggests that you have significant genomic DNA contamination. You might want to throw some DNase on the samples (you'll need to repurify the results, likely just with a column).

[Ahmadiut]

In the Past, I had some samples with DNA contamination, but they had a band between the well and 28s RNA. DNA move in the agarose gel, don't they?
My samples have a lot of gum-resins and carbohydrates. Don't you think that the contaminants in the wells are gum-resin or carbohydrates with absorption in 230 nm? Are these kind of contaminates interfere in the process?

[freestile]

I think that you have genomic DNA (the upper mark of each well). I recommend anyway use DNase previously start libraries preparation. About quality I can't see the the value of 260/280 ratio (must be near 2).
Regards.

[nucacidhunter]

You RNA quality are good judged by nice rRNA bands (I assume these are from non-leave tissues) but NanoDrop is not accurate for quantification. You may try to use RNA specific dye such as Quant-iT RiboGreen RNA reagent for accurate quantification. Sticky materials in the wells most likely will inhibit or reduce enzymatic reactions efficiency during RNAseq library prep processing by preventing access of enzymes and reagents to RNA. While they may not cause library failure but they will impact the quality of data. Tolerance of such effect will be depending on your experiment aims. A column based post extraction RNA clean up kit can remove those from samples.

[pmiguel]

About quality I can't see the the value of 260/280 ratio (must be near 2).
Regards.

Well you can read the value at 260nm and 280nm as "4.5" and "2.0", respectively. But both of these numbers are meaningless as they reflect a small residual amount of phenol in the sample, not the nucleic acids.

See:

http://seqanswers.com/forums/showthread.php?t=21280

for common optically absorbing contaminants of DNA and RNA preps.

The stuff stuck up in the well may or may not be DNA. It could be RNA complexed with glop that is hindering its migration into the gel. Or DNA, or a mixture of both.

Genomic DNA if you don't beat it up too bad during prepping will tend to be in the 50-150kb range so it might not migrate into your gel much by the time rRNA had migrated as far as it had.

--
Phillip